Process for producing soybean saponin-containing material

ABSTRACT

It is intended to easily separate saponins from isoflavones extracted from starting soybeans thereby giving highly pure saponins at a high yield on an industrial scale. Saponins can be highly efficiently separated from isoflavones by performing multistage extraction under mild conditions such that malonyl isoflavone glycoside, from among isoflavones contained in the starting soybeans, is not converted into isoflavone glycoside, acetyl isoflavone glycoside or isoflavone aglycon. Thus, highly pure saponins can be obtained at a high yield on an industrial scale.

TECHNICAL FIELD

The present invention relates to a soybean saponin-containing materialand a process for producing the same.

BACKGROUND ART

Soybean saponins (hereinafter, simply noted as “saponins”) are a genericname of saponins contained in starting soybeans and are contained insoybean hypocotyls in an amount of about 2 to 4% by weight. Saponins areclassified into group A saponin, i.e., bisdesmoside saponin, wherein anaglycone skeleton is “soyasapogenol A” and sugar chains are attached toC-3 position and C-22 position of the aglycone through ether bonds;group B saponin, i.e., monodesmoside saponin, wherein an aglyconeskeleton is “soyasapogenol B” and a sugar chain is attached to C-3position of the aglycone through ether bond; and the like. In addition,saponin, wherein a moiety of a sugar chain is acetylated is alsoreported (Kitagawa et al, Chem. Phrm. Bull, 33, (1985)).

R1 R2 R3 Group A saponin Sugar Chain Sugar Chain —OH Group B saponinSugar Chain —OH —OH

On the other hand, isoflavones are contained in starting soybeans astrace components other than saponins, and contained in soybeanhypocotyls in an amount of about 1 to 2% by weight. Isoflavones aregeneric name for isoflavone aglycone whose aglycone skeleton isdaidzein, glycitein or genistein; isoflavone glycoside, wherein a sugarchain is attached at position 6 of the isoflavone aglycone; and furtheracetyl isoflavone glycoside or malonyl isoflavone glycoside, wherein itssugar chain has an acetyl group (—COCH₃) or a malonyl group (—COCH₂COOH)as a functional group.

Saponins are said to have a variety of physiological activities such asanti-obesity activity, antioxidant activity, and an immunity activationactivity. Then, when a material containing saponins at a highconcentration is available, the material can be utilized by simplyadding it to various foods and drinks.

Conventionally, in order to obtain a material containing saponins at ahigh concentration from starting soybeans such as whole soybeans,defatted soybeans, soybean hypocotyls, and the like, it has been knownthat appropriate separation of saponins from isoflavones is important(JP 62-5917 A and JP 61-129134 A). Hence, as described in thesegazettes, these processes require carrying out purification by gelfiltration such as LH-20, or the like, or partition chromatography aftercarrying out first stage production steps involving extraction with asolvent such as a lower alcohol or acetone, adsorption of the resultantextract with a synthetic adsorption resin such as HP-20 and XAD-2 oractivated carbon, washing the absorbed extract with a low concentrationof a lower alcohol (10 to 40% by volume) and then elution of a fractioncontaining saponins with a high concentration of a lower alcohol (70 to80% by volume).

However, in order to obtain a highly pure saponin fraction in theabove-described processes, the final purifying step by means of gelfiltration or partition chromatography is required and a multi-stagepurifying step is essential. Hence, a yield of saponins is as low asabout 1 to 2%. Indeed, for example, the yield of saponins disclosed inExample 1 of JP 62-5917 A from starting soybeans is 1.38%. This isproblematic from the viewpoints of facility costs and productionefficiency for industrially putting such a process into practice. On theother hand, in order to extract saponins with a solvent at a high yield,conventionally, the extraction is carried out at a high temperature.However, even when such extraction is carried out, a material containingsaponins which satisfies requirements of high purity and high yieldwithout complicated operation cannot be obtained.

Thus, an object of the present invention is to facilitate separation ofsaponins from isoflavones extracted from starting soybeans to obtain ahighly pure saponin-containing material at a high yield on an industrialscale.

DISCLOSURE OF THE INVENTION

Regarding the above problem, the present inventors have studiedextraction conditions to improve purity and yield of saponins. That is,when the present inventors have studied by raising an extractiontemperature, or changing a pH, a yield of saponins can be improved underhigh temperature conditions, but at the same time solid materials otherthan saponins are also extracted, which conversely causes a decrease inpurity of saponins. Therefore, these conditions require purificationoperation of two complicated stages as in a conventional technique, andthus resulting in a decrease in a yield. Hence, we have decided toinvestigate another process.

Then, we have studied intensively to obtain a highly concentratedsaponin-containing material at a high yield by means of simple operationwithout carrying out multi-stage purification operation, and haveobtained the following findings.

(1) Hydrophobicities of saponins are particularly close to those ofisoflavone glycoside, acetyl isoflavone glycoside, and isoflavoneaglycone, among isoflavones. Therefore, it is difficult to separate themby utilizing these properties.

(2) Among isoflavones, malonyl isoflavone glycoside has a dissociationgroup and thus has a relatively high polarity. Therefore, malonylisoflavone glycoside can be easily separated from saponins with anadsorbent.

(3) In a natural state, soybeans contain a largest amount of malonylisoflavone glycoside. However, malonyl isoflavone glycoside is unstableto heat and is easily converted into isoflavone glycoside, acetylisoflavone glycoside, or isoflavone aglycone by heating.

From the above findings, when multi-stage extraction is carried outunder mild conditions at which conversion of malonyl isoflavoneglycoside in starting soybeans into isoflavone glycoside, acetylisoflavone glycoside, or isoflavone aglycone is avoided, saponins can beseparated from isoflavones extremely efficiently only by subsequentone-stage purification step, i.e., treatment with an adsorbent to obtainhighly pure saponins at a high yield, which leads to a finding of asimple and easy process for producing a saponin-containing materialhaving an extremely high purity at a high yield on an industrial scale.Thus, the present invention has been completed.

That is, the present invention discloses the following:

1. A process for producing a soybean saponin-containing material whichcomprises preparing a water-containing polar organic solvent extractsatisfying the following conditions (a) and (b) from starting soybeans:

-   -   (a) a content of malonyl isoflavone glycoside being 25% by        weight or more based on the total amount of isoflavones in said        extract, and    -   (b) an extraction ratio of soybean saponins from said starting        soybeans being 60% by weight or more;

2. The process for producing a soybean saponin-containing materialaccording to the above 1, wherein said starting soybeans are raw soybeanhypocotyls;

3. The process for producing a soybean saponin-containing materialaccording to the above 1 or 2, wherein a water content of saidwater-containing polar organic solvent is from 20 to 85% by volume;

4. The process for producing a soybean saponin-containing materialaccording to any one of the above 1 to 3, wherein extraction is carriedout at a temperature of 10 to 72° C.;

5. The process for producing a soybean saponin-containing materialaccording to any one of the above 1 to 4, wherein extraction is carriedout in a multi-stage manner;

6. A process for producing a soybean saponin-containing material whichcomprises purifying the water-containing polar organic solvent extractprepared by the process according to any one of the above 1 to 5 bytreatment with an adsorbent;

7. A process for producing a soybean saponin-containing material whichcomprises subjecting the water-containing polar organic solvent extractprepared by the process according to any one of the above 1 to 5 totreatment with an adsorbent; eluting a soybean isoflavone-containingfraction with a water-containing polar solvent having a water content of65 to 90% by volume; and then eluting a soybean saponin-containingfraction with a water-containing polar solvent having a water content of5 to 40% by volume;

8. A soybean saponin-containing material obtained by the processaccording to the above 7.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be illustrated in detail.

The present invention is characterized in that, in steps for preparing asaponin-containing extract (hereinafter, noted as a “water-containingpolar organic solvent extract”) from starting soybeans with awater-containing polar organic solvent, the extraction is carried outsuch that a content of malonyl isoflavone glycoside is 25% by weight ormore, preferably 40% by weight or more, more preferably 50% by weight ormore based on the total amount of isoflavones in the extract, and anextraction ratio of saponins in the water-containing polar organicsolvent is 60% by weight or more, preferably 70% by weight or more, morepreferably 80% or more. When the extraction is carried out under theseconditions, highly pure saponins can be obtained at a high yield on anindustrial scale. For obtaining a water-containing polar organic solventextract which meets these conditions, production conditions such asstarting soybeans, a kind and a water content of the water-containingpolar organic solvent, an extraction temperature, an extraction pH, andan extraction method are appropriately combined. Hereinafter, apreferred aspect for obtaining the water-containing polar organicsolvent extract that satisfies the above conditions will be illustrated.

[Process for Preparing Water-Containing Polar Organic Solvent Extract]

(Starting Soybeans)

In the present invention, as a raw material for extracting saponinstherefrom, saponin-containing starting soybeans such as whole soybeans,dehulled soybeans, dehulled and hypocotyl-removed soybeans, defattedsoybeans, soybean hypocotyls, or the like can be used. In particular,soybean hypocotyls having a relatively high saponin content (about 2 to4% by weight) as well as a high content of group A saponin which is saidto have particularly strong antioxidant activity among saponins(Ohminami et al, Proc. Symp. WAKAN-YAKU, 14, 157-162 (1981)) ispreferably used from the viewpoint of a yield. In addition, soybeanprocessed products such as soybean protein isolate, “okara (insolubleresidue from soybean milk production, also known as soy pulp)”, and tofucan also be used. As soybean hypocotyls, those having less hulls andhighly pure hypocotyls are suitable.

Soybean hypocotyls may also be subjected to pre-treatment such as dryheating or wet heating to improve flavor thereof and inactivate trypsininhibitor. However, when treatment is carried out to such extent thatmalonyl isoflavone glycoside is converted into isoflavone glycoside andacetyl isoflavone glycoside, separation efficiency in the purificationstep after extraction can be adversely affected. Therefore, raw soybeanhypocotyls are preferably used. When pre-treatment is carried out, mildtreatment wherein conversion of malonyl isoflavone glycoside isminimized is preferred.

Examples of the isoflavone compositions of raw soybean hypocotyls andsoybean hypocotyls subjected to dry heating are shown in Table 1. Inthese examples, malonyl isoflavone glycoside in soybean hypocotylssubjected to dry heating (roasting) is mostly converted into isoflavoneglycoside or acetyl isoflavone glycoside, and therefore dry heating tosuch extent is not preferred.

TABLE 1 Raw Dry heated Isoflavone composition hypocotyls hypocotylsIsoflavone glycosides Daidzin 10.9 25.5 Genistin 2.7 6.7 Glycitin 8.719.5 Malonyl isoflavone glycosides Malonyl daidzin 48.7 0.1 Malonylgenistin 9.7 0.1 Malonyl glycitin 18.2 0.3 Acetyl isoflavone glycosidesAcetyl daidzin 0.2 21.8 Acetyl genistin 0.0 7.5 Acetyl glycitin 0.2 15.3Isoflavone aglycons Daidzein 0.4 0.6 Genistein 0.0 0.2 Glycitein 0.3 2.4Total 100.0 100.0

(Extraction Solvent)

As a solvent for extracting saponins from starting soybeans, awater-containing polar organic solvent, i.e., an organic solventmiscible with water which contains water in a desired ratio is used.Examples of a hydrophilic organic solvent include lower alcohols such asmethanol, ethanol, propanol, etc.; acetone; and the like. In particular,ethanol is preferably used. It is advisable to avoid the use ofnon-polar organic solvents such as ether, chloroform, hexane, etc.,because they have inferior extraction efficiencies of glycosidecomponents such as isoflavones and saponins.

A water content of the water-containing polar organic solvent variesdepending upon the kind of a polar organic solvent and may be selectedso that highly pure saponins can be extracted at a high yield. The watercontent is preferably 20 to 85% by volume, more preferably from 25 to70% by volume, still more preferably from 25 to 60% by volume. When thewater content is too small, a saponin extraction ratio is extremelylowered. On the other hand, when it is too large, saponins cannot beseparated from isoflavones easily and purity of saponins tends to belowered due to an increase in a ratio of isoflavone aglycons formed bydecomposition of isoflavone glycoside and its malonyl and acetylderivatives, perhaps, because of β-glycosidase present in soybeanhypocotyls.

The amount of the water-containing polar organic solvent relative tostarting soybeans to be used for each extraction is not specificallylimited, and varies depending upon the kind of a solvent. From aneconomical viewpoint, preferably, the amount is 3 to 10 L per 1 kg ofstarting soybeans.

(Extraction Temperature)

Extraction of saponins from starting soybeans is preferably carried outat such a temperature that malonyl isoflavone glycoside is hardlyconverted into isoflavone glycoside and acetyl isoflavone glycoside, orlower, more preferably from 10 to 72° C., still more preferably from 20to 65° C. When the extraction temperature is too low, saponins cannot beextracted easily and a yield tends to be lowered. On the other hand,when the extraction temperature is too high, saponins cannot beseparated easily and purity of saponins tends to be lowered due to anincrease in isoflavone glycoside and acetyl isoflavone formed byhydrolysis or decarboxylation of the malonyl group of malonyl isoflavoneglycoside.

(Extraction pH)

The water-containing organic solvent is preferably pH 4 to 9, morepreferably 5 to 8. When the pH is too low, saponins in the resultantextract tends to precipitate depending upon a water content of thewater-containing organic solvent, thereby making the subsequentfractionation step troublesome. On the other hand, when the pH is toohigh, separation efficiency of saponins from isoflavones tends to belowered because malonyl isoflavone glycoside is easily converted intoisoflavone glycoside.

(Extraction Method)

An extraction method may be selected so that an extraction ratio ofsaponins from starting soybeans into the water-containing polar organicsolvent extract is 60% by weight or more, more preferably 70% by weightor more, still more preferably 80% by weight or more. For example, it ispreferable to select a multi-stage extraction method wherein at leasttwo or more-stage extraction is carried out. In this procedure,extraction may be carried out plural times in one extraction vessel(tank) (butch-wise), or once or more in each of plural tanks connectedto each other in series. Further, it is also possible to arrange one ormore tanks in parallel to each of tanks connected to each other inseries. In addition to a multi-stage extraction method, a counterflowcontinuous-extraction method or the like can also be employed. Themethods illustrated above can be selected by taking into account thespace in a production site, costs, and the like. On the other hand, incase that the extraction is carried out only once, an extraction ratioof saponins from starting soybean cannot be increased easily and a yieldtends to be lowered.

In the water-containing polar organic solvent extract obtained from theabove aspect, saponins are recovered from starting soybeans at a highyield (60% by weight or more), and the ratio of malonyl isoflavoneglycoside in total isoflavones is high (25% by weight or more). Theextract satisfying such conditions makes the subsequent separation ofsaponins from isoflavones and purification of saponins extremely easy.On the other hand, when the extraction is not carried out under mildconditions, and the content of malonyl isoflavone glycoside becomes lessthan 25% by weight based on the total isoflavones in the resultantwater-containing polar organic solvent extract, and conversely thecontent of isoflavone glycoside, acetyl isoflavone glycoside, andisoflavone aglycone is increased to 75% by weight or more, theseparation of saponin-containing fraction therefrom becomes difficult,which extremely lowers purity of saponins, and requires a complicatedpurification step.

[Purification Method of Saponin-Containing Fraction]

Then, an isoflavone fraction can be extremely easily removed from theresultant water-containing polar organic solvent extract by means of aconventional purification means to obtain a highly pure saponin fractionat a high yield. Hereinafter, a preferred aspect of the purificationstep will be illustrated.

(Treatment of Extract with Adsorbent)

The resultant water-containing polar organic solvent extract is firsttreated with an adsorbent. That is, saponins are adsorbed on anadsorption resin or the like. As the adsorption resin, a well-knownresin can be used. Preferred examples to be used include a syntheticadsorption resin such as a porous styrene-divinylbenzene type resin, forexample, HP-20 (manufactured by Mitsubishi Chemical Corporation), SP-825(manufactured by Mitsubishi Chemical Corporation), Amberlite XAD-2 orXAD-4 (manufactured by Rohm and Haas Company), Duolite S-861 or S-862(manufactured by Sumitomo Chemical Co. Ltd.), and the like. Thisprocedure may be carried out by introducing the resin into a tank inbatch-wise, or can be performed by packing the resin into a column.

(Elution of Isoflavone-Containing Fraction)

Then, a fraction highly containing saponins adsorbed on the resin iseluted to purify a saponin-containing fraction. Specifically, thepurification can be carried out by the following method.

Components other than saponins, e.g., isoflavones and the like areselectively removed from the fraction adsorbed on the resin with awater-containing polar organic solvent having a high water content. Thewater content of the water-containing polar organic solvent ispreferably from 65 to 90% by volume. When the water content is too high,a large amount of a solvent is required to elute an isoflavone fraction.On the other hand, when the water content is too low, saponins as wellas isoflavones are eluted simultaneously, which lowers the separationefficiency, thereby lowering the purity of saponins.

(Elution of Saponin-Containing Fraction)

Then, a saponin-containing fraction is selectively eluted with awater-containing polar organic solvent having a low water content andrecovered. The water content of the water-containing polar organicsolvent is preferably 5 to 40% by volume, more preferably 10 to 35% byvolume. When the water content is too high, not only a large amount of asolvent is required to elute a saponin-containing fraction, but alsosaponins cannot be recovered completely, thereby causing a low yield. Onthe other hand, when the water content is too low, efficiency of elutionis lowered, thereby increasing costs.

(Processing of Saponin-Containing Fraction)

An eluent of the resultant saponin-containing fraction as such can beprocessed into a concentrated solution, or processed into powders orgranules to obtain the “soybean saponin-containing material.”

The resultant soybean saponin-containing material has high purity suchas that having a saponin content of 40% by weight or more, preferably50% by weight based on the solid matter. In addition, a yield fromstarting soybeans is high. Thus, the material is a product obtained by aprocess of remarkably high production efficiency.

In case of using soybean hypocotyls as starting soybeans, group Asaponin is contained in an amount of 60% or more in the resultantsaponin-containing fraction. Therefore, such a product is very useful asthe saponin-containing material having higher antioxidant activity.

(Application to Foods, Etc.)

By ingestion of the resultant soybean saponin-containing material,anti-obesity effect, antioxidant effect, lever damage-improving effect,serum lipid-improving effect, and the like are also expected. Then, thematerial can be utilized for tablets, confectionary, drinks, otherhealth drinks and foods, medical supplies, cosmetics, and the like.

In the present invention, saponins were determined by thin layerchromatography. And, isoflavones were determined according to SoybeanIsoflavone Food Specification Standard Analysis Method of Japan HealthFood & Nutrition Food Association. Hereinafter, each method will beillustrated.

(Method for Determination of Saponins)

A sample was precisely weighed, and methanol was added thereto. Afterstirring and extracting for one hour, the mixture was centrifuged toobtain an extract. This was repeated again and the combined extract wasmade up to a given volume. This was applied to thin layer chromatography(TLC), and then the Rf values of the sample were compared with those ofsaponin standards to confirm the spots of saponins. By using a standardstraight line prepared with saponin standards in advance, the areaintegration values of the spots were measured to calculate the amount ofsaponins. Table 2 shows the conditions of thin layer chromatography.

TABLE 2 TLC Conditions TCL plate Silica gel 60F245 (0.25 mm thick,manufactured by Merck Corp.) Developing chloroform-methanol-water mixedsolution solvent (65:25:10, v/v/v lower layer) Development 18 cmDetection Ten (10)% sulfuric acid solution was sprayed, and then heatedat 105° C. for 15 min.

(Method for Determining an Isoflavone)

A sample containing 1 to 10 mg of soybean isoflavones was preciselyweighed, and 25 mL of 70% by volume ethanol was added thereto. Afterstirring and extracting at room temperature for 30 minutes, the mixturewas centrifuged to obtain an extract. Further, the residue was subjectedto similar extraction operation twice. The resultant three extracts werecombined, made up to 100 mL with 70% by volume ethanol, and thenfiltrated with a 0.45 μm PVDF filter to obtain a test solution. Theidentification testing of soybean isoflavones was carried out by using12 kinds of the standards, i.e., daidzin, genistin, glycitin, daidzein,genistein, glycitein, malonyl daidzin, malonyl genistin, malonylglycitin, acetyl daidzin, acetyl genistin, and acetyl glycitin (WakoPure Chemical Industries, Ltd.) to confirm peaks having almost the sameretention times. The determination testing was carried out bydetermining isoflavone concentrations (in terms of daidzin values) of 12kinds with the daidzin standard and true isoflavone concentrations werecalculated by multiplying the determination coefficients below.

Determination coefficients of isoflavones: daidzin (1.000), genistin(0.814), glycitin (1.090), malonyl daidzin (1.444), malonyl genistin(1.095), malonyl glycitin (1.351), acetyl daidzin (1.094), acetylgenistin (1.064), acetyl glycitin (1.197), daidzein (0.583), genistein(0.528), glycitein (0.740).

The amount of isoflavones was calculated from the total of respectiveisoflavone concentrations.

HPLC conditions of the test solutions and the standard solutions areshown in Table 3.

TABLE 3 HPLC Conditions Column YMC-Pack ODS-AM-303 (4.6 × 250 mm) Mobilephase A solution acetonitrile:water:acetic acid = 15:85:0.1 (v/v/v) Bsolution acetonitrile water:acetic acid = 35:65:0.1 (v/v/v) A solutionto linear concentration gradient B solution (for 50 min) Flow rate 1.0ml/min Temperature 25° C. Detection UV 254 nm Injection 10 μL amount

EXAMPLES

The following Examples further illustrate the present invention indetail but are not to be construed to limit the technical scope thereof.In the Examples, all “percents” are by weight unless otherwise stated.

Example 1

To 500 g of raw soybean hypocotyls was added 2.0 L of water-containingethanol having a water content of 25% by volume and the resultantmixture was stirred and extracted at 40° C. After separation of theextract by filtration, again thereto was added 2.0 L of water-containingethanol having a water content of 25% by volume, and the resultantmixture was similarly extracted. The resultant two extracts werecombined (the resultant extract was referred to as a “water-containingpolar organic solvent extraction solution”). The extract was condensedat 40° C. under reduced pressure.

The extract thus obtained was dissolved in water and the resultingsolution was applied to a column (100 ml) packed with a porousstyrene-divinylbenzene type synthetic adsorption resin Diaion HP-20(manufactured by Mitsubishi Chemical Corporation) at SV2.

Then, the column was washed with water-containing ethanol having a watercontent of 80% by volume and further eluted with water-containingethanol having a water content of 20% by volume to obtain a saponinfraction. This fraction was condensed at 40° C. under reduced pressureto obtain a saponin-containing material in the form of a dry powder.

Example 2

According to the same manner as that described in Example 1, asaponin-containing material was obtained except that extraction of rawsoybean hypocotyls with water-containing ethanol was carried out at 70°C.

Example 3

According to the same manner as that described in Example 1, asaponin-containing material was obtained except that extraction of rawsoybean hypocotyls with water-containing ethanol was carried out at 15°C.

Comparative Example 1

According to the same manner as that described in Example 1, asaponin-containing material was obtained except that the extraction wascarried out only once.

Comparative Example 2

According to the same manner as that described in Example 1, asaponin-containing material was obtained except that the startingsoybeans were changed to roasted soybean hypocotyls.

Table 4 shows saponin yields from starting soybeans (% by weight), andabundance ratios (%) of malonyl isoflavone glycoside (Mal-iso), acetylisoflavone glycosides (Ac-iso), isoflavone glycosides (Iso) andisoflavone aglycons (Agl) by taking the total amount of isoflavones ineach extract as 100% of the respective water-containing polar organicsolvent extracts obtained in Examples 1 to 3 and Comparative Examples 1and 2; as well as saponin contents (% by weight), isoflavone contents (%by weight) and saponin yields from starting soybeans of the respectivesaponin-containing materials obtained by treatment with the adsorbent.

TABLE 4 Comparative Comparative Example 1 Example 2 Example 3 Example 1Example 2 (Extraction conditions) Starting raw hypocotyls raw hypocotylsraw hypocotyls raw hypocotyls Roasted Soybeans hypocotyls Extraction 40°C. 70° C. 15° C. 40° C. 40° C. temperature Number of 2 2 2 1 2extractions Water content 25% 25% 25% 25% 25% (Water-containing polarorganic solvent extract) Saponin yield 85% 88% 68% 42% 82% Isoflavoneabundance ratio (%) Mal-iso 54.2 25.3 59.6 47.0 0.5 Ac-iso 4.3 6.2 4.32.3 45.0 Iso 39.5 68.5 33.5 49.2 51.0 Agl 2.0 0.0 2.6 1.5 3.5(Saponin-containing material) Saponin content 60% 40% 56% 48% 25%Isoflavone  2%  8%  6%  3% 18% content Saponin yield 72% 73% 56% 32% 78%

As seen from Table 4, in Example 1 the saponin yield of the extract wasas extremely high as 85% and the abundance ratio of malonyl isoflavoneglycoside was as high as 54.2%. As a result, in the saponin-containingmaterial obtained, the saponin content was extremely high (60%), and theyield was also high (72%). On the other hand, the isofravone content wasonly 2% by weight, indicating that extremely appropriate separation ofisoflavones and saponins from the extract was accomplished, though,usually, separation of isoflavones and saponins was difficult. Further,67% of saponins contained in the saponin-containing material in Example1 was group A saponin.

In Example 2, the saponin yield of the saponin-containing material wasextremely high (73% by weight) as in Example 1, but the abundance ratioof malonyl isoflavone glycoside of the extract was 25.3%, therebylowering the saponin content of the saponin-containing material ascompared with that for Example 1. Nevertheless, the saponin content was40% by weight and this was a satisfactory content. This lowering of thesaponin content to 25.3% is considered to be due to the extractiontemperature of 70° C., which causes lowering of the abundance ratio ofmalonyl isoflavone glycoside in the extract as compared with that ofExample 1, while increasing the abundance ratios of acetyl isoflavoneglycoside and isoflavone glycoside.

On the other hand, in Comparative Example 1, although the saponincontent of the resultant saponin-containing material was 48% by weight,the saponin yield of the extract was 42% by weight, probably, due to theonly once extraction. As a result, the saponin yield of thesaponin-containing material was as low as 32% by weight, therebylowering the production efficiency.

In Comparative Example 2, although the saponin yield of thesaponin-containing material was high (78% by weight), while the saponincontent of the saponin-containing material was as extremely low as 25%by weight. Further, the content of isoflavones was 18% by weight. Thisshowed that the fractionation of isoflavones and saponins was notappropriately carried out. This is considered to be caused by the use ofdry heated (roasted) soybean hypocotyls as starting soybeans, therebycausing conversion of heat unstable malonyl isoflavone glycoside intoacetyl isoflavone glycoside.

Consequently, it has been found that the higher an abundance ratio ofmalonyl isoflavone glycosides of an extract is, the better saponins arefractionated. Further, it has been found that, as the abundance ratio ofmalonyl isoflavone glycosides is decreased and as the abundance ratiosof acetyl isoflavone glycoside and isoflavone glycoside are increased,elution patterns of isoflavones and saponins during the separation withan adsorption resin are overlapped with each other, making it difficultto separate isoflavones from saponins.

Experiment 1

Study of Water Content of Water-Containing Polar Organic Solvent

In order to study the influence of an water content of awater-containing polar organic solvent during extraction, variations ofcontents of saponins and isoflavones in extracts obtained according tothe same manner as that in described Example 1 were examined by changingwater contents of extraction solvents as shown in Table 5.

TABLE 5 Water content (%) 0 10 30 50 60 80 90 (Isoflavone Saponin yield(%) abundance ratio) 10 38 86 82 82 76 63 Mal-iso 28.6 45.3 55.3 59.660.3 65.3 18.0 Ac-iso 0.0 0.0 3.3 4.1 6.3 2.1 6.3 Iso 64.3 46.6 36.333.0 31.1 20.6 23.7 Agl 7.1 8.1 5.1 3.3 2.3 12.0 52.0

As seen from Table 5, when the water content was 0% or 10%, the recoveryrate of saponins was inferior. Further, the abundance ratio of malonylisoflavone glycoside tended to be decreased. When the water content was90%, the saponin recovery rate was high, but the abundance ratios ofisoflavone glycoside and malonyl isoflavone glycoside were extremelylowered. It is considered that this is due to decomposition ofglycosides by the action of β-glucosidase to form aglycons. As a result,from the viewpoints of the saponin recovery rate and the abundance ratioof malonyl isoflavone glycoside, the water content of a water-containingpolar organic solvent to be used for extraction is preferably 20 to 85%by volume, more preferably 25 to 75% by volume.

Experiment 2

Influence of pH of Extract

In order to study the influence of pH during extraction, the saponinrecovery rate and the isoflavone abundance ratio in an extract obtainedaccording to the same manner as that described in Example 1 weremeasured by changing the pH of a water-containing polar organic solventas shown in Table 6. The pH was adjusted by using hydrochloric acid anda sodium hydroxide solution.

TABLE 6 Comparative Comparative Example 1 Example 3 Example 4 Extractionsolvent pH 6.5  3.5 11.0 Saponin yield (%) 85 43 68 Isoflavone abundanceratio (%) Mal-iso 54.2 37.9  2.3 Ac-iso 4.3  2.3  4.1 Iso 39.5 49.2 82.8Agl 2.0 10.6  8.8

As shown in Table 6, in Comparative Example 3, a precipitate was formedduring extraction and the saponin recovery rate was extremely lowered.Further, in recovery of the extract, the separation was inferior and theoperation efficiency was extremely decreased. This is considered to becaused by lowering of solubility of saponins due to lowering of pH andfurther by lowering of the saponin recovery rate due to co-precipitationof saponins consequent to formation of a protein precipitate.

In Comparative Example 4, the abundance ratio of malonyl isoflavoneglycoside in the extract was extremely lowered. This is considered to becaused by instability of malonyl isoflavone glycoside under alkalineconditions. The malonyl group is easily dissociated, thereby convertingmalonyl isoflavone into the glycoside. Then, elution patterns ofisoflavones and saponins are overlapped during the separation with anadsorbing resin, which makes it difficult to separate the isoflavonesand the saponins. Thus, it has been judged that a highly pure soybeansaponin-containing material cannot be obtained.

As a result, in view of the saponin content and recovery rate, it isconsidered that the pH of a water-containing polar organic solvent ispreferably 4 to 9, which is nearly neutral, more preferably 5 to 8.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to obtain a highlypure saponin-containing material at a high yield on an industrial scaleonly by such one purification step as treatment with an adsorbentwithout a subsequent multi-stage purification step such as gelfiltration and partition chromatography from a saponin-containingsolution extracted from starting soybeans, thereby providing a processfor producing a saponin-containing material having extremely highproduction efficiency. Therefore, the present invention is extremelyuseful in industrial fields such as foods, cosmetics, and medicalproducts.

1. A process for producing a soybean saponin-containing material whichcomprises the steps of: extracting raw soybean hypocotyls, withoutpre-treatment thereof, with a water-containing polar solvent having awater content of 20 to 85% by volume at 10 to 72° C. by multi-stageextraction to prepare a water-containing polar organic solvent extractsatisfying the following conditions (a) and (b): (a) a content ofmalonyl isoflavone glycoside being 25% by weight or more based on thetotal amount of isoflavones in said extract, and (b) an extraction ratioof soybean saponins from said raw soybean hypocotyls being 60% by weightor more; purifying the water-containing polar organic solvent extractonly by treatment with an adsorbent; eluting a soybeanisoflavone-containing fraction with a water-containing polar solventhaving a water content of 65 to 90% by volume; and then eluting asoybean saponin-containing fraction with a water-containing polarsolvent having a water content of 5 to 40% by volume to obtain thesoybean saponin-containing material.
 2. The process according to claim1, wherein 60% or more of saponins in the saponin-containing material isgroup A saponin.
 3. A method for separation of soybean saponins fromisoflavones which comprises the steps of: extracting raw soybeanhypocotyls, without pre-treatment thereof, with a water-containing polarsolvent having a water content of 20 to 85% by volume at 10 to 72° C. bymulti-stage extraction to prepare a water-containing polar organicsolvent extract satisfying the following conditions (a) and (b): (a) acontent of malonyl isoflavone glycoside being 25% by weight or morebased on the total amount of isoflavones in said extract, and (b) anextraction ratio of soybean saponins from said raw soybean hypocotylsbeing 60% by weight or more; purifying the water-containing polarorganic solvent extract only by treatment with an adsorbent; eluting asoybean isoflavone-containing fraction with a water-containing polarsolvent having a water content of 65 to 90% by volume; and then elutinga soybean saponin-containing fraction with a water-containing polarsolvent having a water content of 5 to 40% by volume to obtain thesoybean saponin-containing material.